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BIOLOGY 


THE  UNIVERSITY  OF  CHICAGO 


iE  PREPARATION  OF  BACTERIAL 
ANTIGENS 

A  DISSERTATION 

SUBMITTED  TO  THE  FACULTY  OF  THE  OGDEN  GRADUATE 

SCHOOL   OF  SCIENCE   IN    CANDIDACY  FOR  THE 

DEGREE  OF  DOCTOR  OF  PHILOSOPHY 

DEPARTMENT  OF  ANATOMY 

FROM  THE  LABORATORY  OF  PREVENTIVE 
MEDICINE 

BY 

CLARK  OWEN   MELICK 


CHICAGO,   ILLINOIS 

JUNE,   1922 


Q 


THE  PREPARATION  OF  BACTERIAL  ANTIGENS  * 

C.  O.  MELICK,  PH.D. 
(From  the  Laboratory  of  Preventive  Medicine,  University  of  Chicago.) 

The  power  of  the  so-called  "antibodies"  to  react  selectively 
with  the  protein  antigens  employed  in  their  production,  occa- 
sioned the  early  use  of  these  "bodies"  as  reagents  in  the  de- 
tection of  differences  or  similarities  existing  between  two  or 
more  proteins.  In  extension,  the  method  was  also  early  applied 
to  define  the  closeness  of  the  relationship  between  cells,  as  dis- 
played by  the  degree  of  the  similarity  of  their  constituent 
proteins. 

This  application  was  most  extensively  made  in  the  field  of 
bacteriology  and  has  allowed  a  biological  classification  of  bac- 
teria more  extensive  than  that  afforded  by  differences  in  mor- 
phology, growth  and  pathogenicity.  The  extent  of  this  use 
of  the  antibody-antigen  reaction  has  led  to  a  multiplicity  of 
modifications  of  the  method  first  employed.  But  although 
these  modifications  have  in  general  been  directed  toward  a  re- 
finement and  possible  standardization  of  the  technic,  certain 
phases  remain  relatively  crude  and  conducive  to  error  both  in 
experiment  and  in  interpretation.  Two  distinct  complications 
in  the  methods  now  practised  are;  first,  the  tremendous  ad- 
mixture of  non-essential  substances  complicating  the  condi- 
tions of  reaction  both  physically  and  chemically;  and,  secondly, 
the  chemical  instability  of  the  essential  reacting  substances. 

It  is  the  purpose  of  this  paper  to  discuss  these  complicating 
factors  in  the  antibody-antigen  reaction  and  to  outline  methods 
which  I  have  used  successfully  in  eliminating  such  complica- 
tions. 

*  Received  for  publication  June  14,  1922. 

3 


520-130 


4  MELICK 

The  experimental  work  which  is  the  basis  of  these  studies 
was  performed  in  the  differentiation  of  strains  of  the  colon- 
typhoid-intermediate  group  of  bacteria  by  means  of  the  spe- 
cific antibody-antigen  reaction  as  displayed  in  the  fixation  of 
the  complement  of  a  hemolytic  complex. 

The  results  to  be  presented  in  detail  have  to  do  with  the  pro- 
duction of  standard  antigens,  and  fall  under  three  general 
headings:  first,  the  elimination  of  admixed  culture  medium 
proteins;  second,  the  disintegration  of  the  bacterial  cells  with 
a  minimal  chemical  modification  and  a  maximal  liberation  of 
their  antigenic  proteins;  third,  the  conservation  of  antigens 
without  the  addition  of  chemical  preservatives. 

I.  The  elimination  of  admixed  culture  medium  proteins. 
—  Commonly,  the  bacteria  used  in  the  preparation  of  antigens 
have  been  grown  either  in  protein  containing  liquid  media, 
such  as  meat  infusion  broth,  or  on  the  surface  of  solid  media 
impregnated  with  protein  constituents.  For  members  of  the 
intestinal  group  of  bacteria,  beef  infusion  agar  has  been  most 
commonly  used.  Whole  blood,  blood-serum,  ascites  fluid,  egg, 
and  other  substances  have  been  added  to  the  nutrient  agar 
when  the  organisms  required  special  conditions  for  maximum 
growth.  In  each  case,  protein  substances  have  been  intro- 
duced into  the  medium,  including  in  most  instances  bacterial 
proteins  derived  from  the  massive  number  of  organisms  which 
develop  during  the  commercial  preparation  of  the  so-called 
peptones. 

The  removal  of  bacteria  from  such  a  medium  is  accompanied 
by  a  transfer  of  considerable  quantities  of  the  culture  medium 
constituents  which  cannot  be  entirely  removed  from  the  bac- 
teria even  by  combined  nitration  and  centrifugalization.  The 
use  of  such  an  antigen  is  untrustworthy.  Its  injection  may  well 
stimulate  the  production  of  immune  bodies  to  the  admixed 
culture  medium  proteins  as  well  as  to  the  proteins  of  the  bac- 
teria themselves.  The  resulting  immune  serum  therefore,  when 
tested  with  similarly  prepared  antigens,  may  react  not  only 
with  the  bacterial  proteins  but  also  with  a  variety  of  others 
derived  from  the  medium  itself. 


PREPARATION  OF  BACTERIAL  ANTIGENS  5 

Olitsky  and  Bernstein,1  in  their  studies  of  such  reactions, 
found  that  the  injection  of  serum-grown  bacteria  into  animals 
resulted  in  the  production  of  a  precipitating  serum  versus  the 
serum  present  in  the  medium.  The  antiserum  thus  formed 
reacted  with  suspensions  of  other  species  of  bacteria  grown  on 
the  same  serum  medium,  in  regard  to  precipitation,  agglutina- 
tion, and  complement  fixation.  Their  obvious  conclusion  was 
that  the  bacteria  themselves  were  the  carriers  of  the  serum 
proteins -of  the  culture  medium  in  sufficient  quantities  to  func- 
tion as  complicating  antigens. 

In  a  later  report,2  the  same  authors  were  able  to  show  that 
when  an  immune  serum  was  developed  by  injections  of  bac- 
teria grown  on  "serum"  medium  and  was  inoculated  into 
guinea  pigs,  it  sensitized  the  cells  of  the  animal  not  only  to  the 
bacteria  but  also  to  the  protein  present  in  the  medium  upon 
which  the  bacteria  were  grown. 

These  authors  are  of  the  opinion  that  in  addition  to  the 
traces  of  culture  medium  proteins  which  are  carried  over  as  an 
admixture,  certain  of  the  serum  proteins  of  the  medium  are 
actually  absorbed  by  the  bacterial  cells. 

While  I  do  not  care  to  discuss  this  latter  conclusion,  the 
results  themselves  demonstrate  the  necessity  of  excluding  as 
far  as  possible  from  bacterial  antigen  preparations  all  proteins 
which  are  not  actual  constituents  of  the  bacteria  themselves. 
I  am  convinced  that  the  methods  commonly  used  in  preparing 
bacterial  antigens  are  not  satisfactory  from  this  point  of  view 
and  that  fine  distinctions  based  upon  their  use  are  not  reliable. 

For  these  reasons,  in  my  own  studies  of  the  colon-typhoid- 
intermediate  group  I  sought  to  cultivate  the  organisms  in 
media  containing  no  proteins  and  wish  to  present  here  the 
methods  by  which  this  was  accomplished.  The  actual  an- 
tigenic  variations  of  the  organisms  so  grown  as  regards  com- 
plement fixation  at  periodic  intervals  are  the  basis  of  a  future 
publication. 

Comparative  tests  of  a  number  of  non-protein  synthetic 
media  resulted  in  favor  of  a  modification  of  Uschinsky's  me- 
dium, in  the  preparation  of  which  especial  attention  was 
given  to  the  purity  of  the  reagents  and  to  the  final  degree  of 


O  MELICK 

acidity.    The  medium  as  finally  standardized  is  prepared  by 
the  following  formula: 

Redistilled  Water 1000. 

Sodium  Chloride 5. 

Asparagin 3.4 

Di-Sodium  Hydrogen  Phosphate 2. 

Magnesium  Sulphate .5 

Ammonium  Lactate 6.3 

After  dissolving,  the  reaction  of  the  medium  is  made  +0.2 
acid  by  the  addition  of  sodium  carbonate,  then  autoclaved  for 
ten  minutes  at  ten  pounds  pressure,  tubed  and  again  sterilized 
as  before.  Any  tubes  showing  the  presence  of  a  precipitate 
are  discarded. 

Employing  this  medium  I  made  daily  transfers  for  from  one 
to  two  weeks.  At  the  end  of  this  time,  all  (except  a  very  few) 
of  the  one  hundred  strains  with  which  I  worked  grew  suffi- 
ciently in  forty-eight  to  seventy-two  hours  to  be  used  in  the 
preparation  of  an  antigen.  (The  few  cultures  which  grew 
sparingly  or  not  at  all  when  first  inoculated  in  this  medium 
later  grew  sufficiently  by  first  inoculating  them  into  tubes 
which  contained  1/20  part  broth  in  addition  to  the  protein  free 
medium.  These  cultures  were  transferred  daily  to  tubes  con- 
taining decreasing  amounts  of  the  broth  and  finally  after 
several  transfers  the  cultures  grew  well  in  the  medium  without 
broth  addition.) 

With  the  fact  established  that  an  extensive  cultivation  of  the 
organism  could  be  accomplished  in  this  protein-free  medium, 
a  series  of  experiments  was  next  instituted  to  determine 
whether  or  not  the  antigenic  properties  of  the  bacteria  differed 
when  grown  in  the  protein-free  and  in  the  protein-containing 
media. 

For  this  determination  twelve  rabbits  were  used,  six  of  which 
were  injected  individually  with  B.  typhosus,  B.  paratyphosus 
A,  B.  paratyphosus  B,  B.  enteritidis,  B.  suipestifer,  and  B.  coli 
communis,  grown  on  plain  agar.  The  other  six  were  also  in- 
jected with  the  same  organisms  which  had  been  grown  in  the 
protein-free  medium.  For  injection,  the  organisms  were  killed 
by  heating  at  60°  C.  for  one  hour,  control  tests  being  made  as 
to  non-viability.  An  equal  number  of  organisms  from  each 


8  MELICK 

culture  (as  determined  by  Wright's  method)  were  suspended 
in  i  c.c.  of  NaCl  solution. 

After  the  withdrawal  of  10  c.c.  of  blood  from  each  animal  for 
normal  serum  controls,  the  twelve  rabbits  were  injected  at 
five  day  intervals.  The  first  and  second  injections  were  made 
subcutaneously,  the  third  and  fourth  intraperitoneally  and  the 
fifth,  sixth,  and  seventh  intravenously.  For  test  purposes 
10  c.c.  of  blood  was  drawn  six  days  after  the  last  injection  and 
at  successive  monthly  intervals,  and  in  each  instance  the  serum 
so  obtained  was  titrated  for  its  agglutinating  value  on  the  day 
following  the  bleeding. 

These  determinations  showed  that  the  serum  from  the 
rabbits  which  were  injected  with  the  organisms  grown  in  the 
protein-free  medium  contained  as  much  specific  antibody  as 
did  the  serum  from  the  animals  which  received  organisms 
grown  on  the  protein-containing  medium,  even  when  titrated 
against  organisms  grown  on  the  latter  medium.  The  first 
graph  (Table  I)  displays  the  curves  representing  the  average 
titer  of  each  of  the  two  groups  of  animals  at  different  points. 

From  this  graph  it  can  be  seen  that  at  no  time  was  there 
a  distinct  contrast  in  antibody  content  in  the  sera  of  the  two 
groups  of  animals. 

A  detailed  presentation  of  the  titer  of  the  several  sera  at  the 
time  of  maximum  observed  antibody  content  (six  days  follow- 
ing last  injection)  is  given  in  Table  II  where  the  results  are 
shown  for  each  serum  as  tested  with  the  homologous  organism 
grown  in  both  types  of  culture  medium. 

From  the  results  found  in  Table  II  it  is  apparent  that  with 
the  groups  of  organisms  under  consideration,  not  only  can 
they  be  cultivated  satisfactorily  in  the  absence  of  admixed 
proteins,  but  that  when  so  cultivated  they  display  the  same 
bacterial  antigenic  values  which  the  organisms  possess  when 
grown  in  protein-containing  media. 

II.  The  disintegration  of  bacterial  cells  with  a  minimal 
chemical  modification  and  a  maximal  liberation  of  their  anti- 
genic proteins.  —  While  the  simple  saline  suspensions  of  bac- 
teria as  used  by  Bordet  and  Gengou 3  in  their  original  work 


PREPARATION  OF  BACTERIAL  ANTIGENS 


served  the  purpose  of  establishing  the  principle  of  complement 
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inhibited  the  action  of  the  complement  as  to  disallow  a  con- 
clusion concerning  a  true  fixation  of  the  complement  in  a 
specific  antibody-antigen  reaction. 

That  the  complication  could  be  minimized  by  the  substitu- 
tion of  extracts  of  the  bacteria  for  a  suspension  of  the  cells 
was  demonstrated  in  a  considerable  series  of  experiments  by 


10  MELICK 

Wassermann  and  Bruck,5  Citron,6  Wassermann,7  and  by 
Leucles.8 

Thus  it  early  became  recognized  that  the  products  of  bac- 
terial disintegration  were  to  be  preferred  to  intact  bacterial 
cells  in  the  preparation  of  antigens  for  test  tube  determinations 
by  the  complement  fixation  method.  Of  the  many  means  used 
in  the  attempt  to  obtain  a  suitable  disintegration,  autolysis  is 
perhaps  the  oldest.  Mechanical  procedures  such  as  grinding 
the  dried  organisms,  either  alone  or  admixed  with  a  cutting 
material  such  as  sand  have  also  been  extensively  employed. 
In  general,  these  processes  are  of  slight  efficiency.  Direct 
treatment  by  chemical  reagents  has  also  been  resorted  to,  the 
organism  often  being  subjected  to  the  action  of  alcohol,  ether, 
chloroform,  antiformin  or  the  proteolytic  ferments.  In  the 
more  recent  attempts  to  obtain  thorough  disintegration,  the 
several  procedures  have  been  combined  as  in  Besredka's 
method  by  Gay,  where  alcoholic  precipitation,  desiccation  and 
grinding  with  sodium  chloride  crystals  are  resorted  to,  in 
order. 

None  of  these  methods  is  more  than  partially  satisfactory  in 
obtaining  favorable  antigen  products  from  bacterial  cells.  In 
the  case  of  the  mechanical  procedures,  the  yield  of  cell-free 
antigen  is  slight.  In  the  case  of  the  chemical  methods  a  modi- 
fication of  the  antigenic  bacterial  proteins  by  the  reagents  em- 
ployed is  sure  to  occur  and  may  in  any  given  instance  be  suffi- 
cient to  change  the  antigenic  properties  of  the  native  proteins. 
This  latter  point,  I  cannot  emphasize  too  strongly;  for  it  has 
been  disregarded  with  great  regularity.  If  there  is  one  general- 
ization which  can  be  made  concerning  antigenic  substances, 
it  is  that  they  are  protein  in  structure;  and  equally  valid  is  the 
observation  that  as  such,  they  are  highly  susceptible  to  modi- 
fication by  chemical  reagents.  The  marked  specificity  of  the 
antibody-antigen  reaction  demonstrates  in  itself  the  delicacy  of 
the  chemical  differences  which  determine  characteristic  anti- 
genic properties.  That  these  differences  are  readily  modified 
by  chemical  reagents  is  shown  by  the  work  of  Pick,9  Kahn 
and  McNeil,10  Perry  and  Kolmer  n  and  others.  There  is  no 
guarantee  that  any  cell  protein  subjected  to  reagents  such  as 


PREPARATION  OF  BACTERIAL  ANTIGENS         II 

referred  to,  may  not  be  profoundly  changed  in  its  structure 
and  methods  which  involve  such  possibilities  are  to  be  avoided. 

The  method  of  choice  for  the  preparation  of  a  bacterial  anti- 
genie  product  is  one  which  liberates  the  characteristic  proteins 
from  the  cell  bodies  in  maximal  amounts  and  with  minimal 
chemical  modification.  I  have  sought  to  accomplish  this  by 
the  use  of  physical  means,  and  to  this  end  have  developed  a 
method  in  which  the  essential  feature  is  the  rapid  freezing  and 
thawing  of  the  bacteria  in  aqueous  suspension.  By  this  method 
large  amounts  of  the  native  antigenic  proteins  are  obtained  in 
solution  and  the  anticomplementary  constituents  of  the  cells 
are  removed.  The  method  is  simple,  and  as  I  have  used  it  with 
the  colon- typhoid-intermediate  group,  is  as  follows: 

The  organisms  are  grown  for  from  forty-eight  to  seventy-two 
hours  in  culture  tubes  containing  the  synthetic  medium  previ- 
ously described,  at  the  end  of  which  time  the  entire  contents 
are  transferred  to  silver  plated  metal  tubes.  (The  tubes  are 
made  in  the  following  manner :  A  hole  f  inch  is  drilled  4!  inches 
deep  in  the  center  of  a  i  inch  diam.  solid  steel  rod.  The  rod  is 
cut  off  at  a  length  of  5  inches  thus  leaving  a  J  inch  wall.  The 
ends  are  rounded  off  and  both  the  inside  and  outside  are 
polished.  The  tubes  are  then  heavily  silver  plated.  In  order 
to  keep  the  tubes  from  bursting  during  the  freezing  process,  a 
tapering  silver  plated  pin  is  placed  in  them.  This  pin  is  4  inches 
long  and  &  inch  in  diameter  at  the  top,  through  which  2 
smaller  pins  are  inserted  at  right  angles  in  order  to  hold  it  in 
the  center  of  the  tube.  After  the  material  is  added  to  the 
tubes,  a  cork  is  placed  in  the  end  and  it  is  sealed  tightly  by 
applying  several  layers  of  adhesive  tape.  A  small  metal  handle 
is  inserted  in  the  cork.  The  tubes  are  immersed  in  a  flask  of 
liquid  air  to  within  i  inch  of  their  tops  for  from  three  to  four 
minutes.  They  are  then  removed  and  the  contents  thawed 
quickly  by  dipping  the  tubes  for  a  few  seconds  at  a  time  in 
boiling  water.  By  shaking  the  tubes  vigorously,  the  material 
in  the  tubes,  if  carefully  watched,  may  be  very  rapidly  thawed 
without  the  temperature  of  the  contents  rising  above  40°  C. 
In  this  way,  a  change  of  temperature  of  more  than  200°  C. 
is  obtained  in  a  period  of  from  four  to  five  minutes. 


12 


MELICK 


This  process  is  repeated  in  rapid  succession  from  twenty  to 
twenty-five  times.  The  contents  of  the  tubes  are  then  centrif- 
ugalized,  and  a  clear  supernatant  fluid  is  obtained  with  a 
compact  sediment  consisting  of  fragments  of  the  disintegrated 
bacterial  cells.  The  supernatant  fluid  is  drawn  off  and  re- 
centrifugalized.  Without  further  treatment  the  resulting  clear 
solution  constitutes  the  antigen  preparation.  It  is  tested  for 
antigenic  and  anticomplementary  properties.  The  sediment 
contains  the  great  bulk  of  anticomplementary  substances  with 

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but  little  antigen  and  is  discarded.  For  completeness,  a  pro- 
tocol of  the  antigenic  and  anticomplementary  titrations  ob- 
tained in  preparing  a  given  antigen  from  B.  paratyphosus  B 
by  the  above  method  are  given  in  Table  III.  The  organism 
"Paratyphosus  B  no.  12"  was  grown  seventy- two  hours  in 
synthetic  medium  at  37°  C. 

This  table  shows  that  the  antigen  preparation  when  titrated 
by  the  complement  fixation  method  displayed  a  very  great 
antigen  content,  in  fact  distinctly  greater  than  did  the  total 
suspension;  on  the  other  hand,  it  completely  lacked  an  anti- 


PREPARATION  OF  BACTERIAL  ANTIGENS         13 

complementary  action  of  its  own.  The  sediment,  on  the  con- 
trary, possessed  almost  the  full  anticomplementary  power  of 
the  original  suspension,  but  showed  only  a  slight  antigenic 
value. 

The  method  is  constant  in  providing  a  satisfactory  antigen 
preparation  without  anticomplementary  action  when  applied 
to  the  group  of  organisms  in  question.  The  only  point  in  the 
method  at  which  special  care  must  need  be  exercised  is  at  the 
time  of  centrifugalization.  The  antigen  solution  must  be  centrif- 
ugalized  until  it  is  perfectly  clear,  otherwise  it  may  still  pos- 
sess a  degree  of  anticomplementary  action. 

How  far  the  method  is  applicable  to  other  types  of  organ- 
isms than  those  here  considered  has  not  been  extensively  de- 
termined. But  that  it  has  a  general  application  seems  prob- 
able from  the  fact  that  in  a  slightly  modified  form  I  have  by 
its  use  succeeded  in  preparing  an  antigen  from  tubercle  bacilli 
with  so  low  an  anticomplementary  action  as  to  allow  its  use  in 
a  reliable  serum  diagnosis  of  tuberculosis  in  man. 

III.  The  conservation  of  antigens  without  the  addition  of 
chemical  preservatives.  —  In  the  preceding  section  I  have 
given  methods  by  which  bacteria  may  be  cultivated  for  use  as 
antigens,  without  protein  admixture  and  by  which  the  essential 
antigenic  substances  may  be  released  from  the  bacterial  cell 
without  modification  by  chemical  reagents. 

A  third  necessity  in  the  comprehensive  use  of  antigens  for 
quantitative  test-tube  determinations  is  the  preservation  of  a 
given  antigen  over  a  relatively  long  period  without  great  loss 
in  antigenic  value  or  marked  increase  in  its  anticomplementary 
action. 

The  usual  method  of  adding  to  antigen  solutions  such  chem- 
ical preservatives  as  phenol  and  trikresol  has  the  distinct  disad- 
vantage of  inviting  progressive  chemical  changes  by  these 
reagents  often  resulting  in  precipitates  with  considerable  anti- 
complementary  power. 

A  far  more  dependable  method  I  have  found  to  be  the  preser- 
vation of  stock  antigens  in  a  frozen  state.  In  this  method,  the 
perfectly  clear  antigen  solution  obtained  by  centrifugalization 


MELICK 


as  described  above,  is  subdivided  into  small  glass  tubes.  Each 
tube  is  filled  to  about  two-thirds  its  capacity  and  sealed.  The 
tubes  are  then  placed  in  a  refrigerating  chamber  maintained 
at  —  1 8°  C.  and  remain  there  until  required  for  use. 

Antigens  so  preserved  are  serviceable  for  a  period  of  at  least 
three  years.  Slight  decrease  in  antigenic  value  does  occur  in 
that  time,  but  it  is  not  of  such  degree  as  to  impair  seriously  the 
antigen  as  a  reagent. 


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PREPARATION  OF  BACTERIAL  ANTIGENS  15 

The  periodic  titration  of  an  antigen  so  preserved  is  displayed 
in  Table  IV. 

As  shown  above,  the  antigen  unit  when  first  tested  was 
.015  c.c.  while  2.5  c.c.  displayed  no  anticomplementary  effect. 
After  six  months,  the  antigenic  unit  was  .02  c.c.  whereas  2.5  c.c. 
still  was  not  anticomplementary.  After  one  year,  the  values 
were  the  same  as  at  six  months.  At  two  years,  the  antigenic 
unit  was  found  to  be  .025  c.c.  and  2.5  c.c.  displayed  a  slight 
anticomplementary  action.  At  three  years,  the  findings  were 
the  same  as  at  two,  except  that  the  anticomplementary  action 
was  slightly  increased.  Throughout  the  whole  period  however, 
the  antigenic  value  decreased  less  than  one  half  and  the  final 
anticomplementary-antigenic  [Ac.-Ag.]  index  was  60,  thus 
affording  an  entirely  satisfactory  antigen  for  complement 
fixation  reactions. 

CONCLUSIONS 

As  stated  at  the  beginning  of  this  paper,  the  work  has  been 
directed  to  the  elimination  of  certain  factors  tending  to  lessen 
the  accuracy  of  bacterial  identification  by  the  antigen-anti- 
body reaction. 

I  conclude  that  the  methods  as  given  above  are  efficient: 

1.  In  eliminating  complicating  non-essential  proteins  de- 
rived from  the  culture  medium. 

2.  In  obtaining  from  the  bacterial  cell  an  increased  amount 
of  its  essential  antigenic  proteins  with  a  minimal  modification 
of  their  chemical  structure. 

3.  In  preserving  antigens  without  desiccation  and  without 
the  addition  of  chemical  reagents. 

BIBLIOGRAPHY 

1.  Jour,  of  Inf.  Dis.,  Aug.,  1916,  xix,  2  and  253. 

2.  Jour,  of  Inf.  Dis.,  Feb.,  1917,  xx,  150. 

3.  Annales  de  1'Inst.  Pasteur,  Paris,  1901,  289. 

4.  Berl.  klin.  Wchnschr.,  1906,  xxxviii,  1243. 

5.  Med.  Klinik.,  1905,  Iv. 

6.  Centralbl.  f.  Bakt,  1906,  xli. 

7.  Berl.  klin.  Wchnschr.,  1907,  i. 

8.  Berl.  klin.  Wchnschr.,  1907,  iii,  68  and  107. 

9.  Kolle  u.  Wassermann,  1913,  i,  708. 

10.  Jour,  of  Immunology,  1918,  iii,  277. 

11.  Jour,  of  Immunology,  1918,  iii,  247. 


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